Only a fraction of the DNA contained in the interphase nucleus of a higher organism in active in the support of RNA synthesis. The objective of this project is to gain insights into the molecular basis of this restriction in gene activity and the events associated with gene activation and repression. This could conceivably involve either alterations in the chromatin templates such as the addition, removal or chemical modification of chromosomal proteins, or alterations in the transcriptional specificity of RNA polymerase. This project is specifically concerned with the role of multiple forms of RNa polymerase and associated regulatory molecules in determining the spectrum of genes transcribed within a differentiated cell. Four distinct species of nuclear DNA dependent RNA polymerase have been identified in Novikoff ascites cells. Each species will be purified and the subunit composition determine by gel electrophoresis in the presence of sodium-dodecyl sulfate. Three protein factors have also been characterized that dramatically stimulate the activity of the various polymerase species. Following the purification of these stimulation factors their mechanism of action will be elucidated. To determine if they confer new transcriptional specificity on the RNA polymerase, the RNA products transcribed by each enzyme in the presence and absence of factor will be analyzed by nucleic acid hybridization competition experiments. The localization of these factors within the cell and their relationship to known non-histone chromosomal proteins will also be determined. It is fundamental to our understanding of the process of development and the control of cellular activity in general to first understand the biochemical mechanisms involved in the regulation of gene activity.